CN101092618B - Method for preparing enzyme of dissolving staphylococcal bacteria, its derivative, and method for preparing the derivative - Google Patents
Method for preparing enzyme of dissolving staphylococcal bacteria, its derivative, and method for preparing the derivative Download PDFInfo
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Abstract
This invention relates to a method for preparing lysostaphin and its derivatives. The method comprises: designing and synthesizing lysostaphin gene, inserting lysostaphin gene into the digestion site of expression vector (plasmid pET), transforming E. coli, screening positive clones, culturing, inducing the expression of target protein, collecting E. coli, lysing, centrifuging, collecting the supernatant, and purifying the supernatant through chromatography. The method has such advantages as simple process, high expression level, simple purification, high yield, soluble product, and no requirement for renaturation, and is suitable for mass production. The lysostaphin derivatives are obtained by modifying lysostaphin with polyethylene glycol, and have such advantages as low immunogenicity and long half value period. The lysostaphin and its derivatives are suitable for treating Staphylococcus infection in clinic.
Description
Technical field
The present invention relates to biological technical field, relate in particular to the preparation method of staphylococcus lysozyme and the preparation method of its derivative and derivative.
Background technology
Staphylococcus (Staphylococcus) is a kind of of gram-positive cocci, be distributed widely in nature, on air, water, soil, feed and some article, the body surface, pharynx nasalis and the enteron aisle that also are present in humans and animals, most not pathogenic, minority can cause that humans and animals causes a disease, wherein the streptococcus aureus virulence is the strongest, often causing the suppurative inflammation of skin, tissue and organ, is the main pathogeny bacterium of diseases such as burn wound infection, acute hepatic failure and haematogenous ephritis.Streptococcus aureus is also taken place simultaneously with G-septicopyemia germ, synergy, serious threat patient's life.But the enterotoxin that streptococcus aureus produces is contaminated food and cause food poisoning also.Nasal cavity carries disease germs and reaches 80～100% among the doctor of hospital and the nurse, and often is Resistant strain, is the important factor of hospital infection.
1940, the infection of streptococcus aureus was effectively controlled in the discovery of penicillin G and use, and 90% golden Portugal bacterium is to the penicillin sensitivity.Nineteen forty-four, Britain has found penicillin-fast golden Portugal bacterium.Along with β-class amides microbiotic is extensive use of clinical, the resistant organism ratio constantly increases.Resistant organism shows resistance by producing β-class Ntn hydrolase decomposition microbiotic.Nineteen sixty, succeed in developing the X-1497, because of it shows good therapeutic action to penicillin resistant gold Portugal bacterium clinically to penicillinase is stable.But along with the use of new antibiotic, methicillin-resistant gold Portugal bacterium (MRSA) ratio constantly increases, and streptococcus aureus accounts for first that all gram-positive coccis infect at present.Methicillin resistant staphylococcus aureus accounts for 75% in the streptococcus aureus.
Vancomycin is the choice drug of treatment MRSA, yet Japan in 1997 just isolates first strain to the drug-fast streptococcus aureus of vancomycin moderate, and vancomycin is 8mg/L to its minimal inhibitory concentration.2002, the U.S.'s reported first drug resistance of vancomycin streptococcus aureus, vancomycin is 32mg/L to its minimal inhibitory concentration.The appearance of drug resistance of vancomycin streptococcus aureus makes infectation of bacteria become very thorny clinical problem once more, U.S. CDC had been formulated " VRSA/VISA detects and the control guide " in 2003, increased the drug resistance of vancomycin detection methods of staphylococcus aureus in the NCCLS drug sensitive test operative norms in 2005, required each laboratory to carry out monitoring the drug resistance of vancomycin bacterium.
In the face of golden Portugal bacterium to traditional antibacterials resistance situation serious situation day by day, it is particularly urgent that the development of new antibiotic preparation seems.Staphylococcus lysozyme has caused people's attention as aureus cell wall lyase.1964, Schindler and Schuhardt found from a strain is numbered imitation staphylococcus (Staphylococcus simulans) culture of NRRL B-2628 first and have separated staphylococcus lysozyme (Lysostaphin).This enzyme is the single chain molecule that 246 amino acid are formed, and molecular weight 27kDa, zinc are necessary cofactor.Amino acid lacks halfcystine in forming, and the ratio of basic aminoacids is higher, and iso-electric point is between 10.4～11.4.Staphylococcus lysozyme is second and the 3rd peptide bond that glycine forms in the hydrolytic bacteria whole cell peptidoglycan crosslinking structure glycine pentapeptide bridging specifically, therefore has the broken wall bacteriolysis.Because glycine pentapeptide bridge crosslinking structure only is present in the aureus cell wall in a large number, it is the widest wherein to distribute with golden Portugal mycetocyte wall again, so staphylococcus lysozyme mainly has the bacteriolyze germicidal action to the especially golden Portugal of staphylococcus bacterium.
The early stage main means that obtain staphylococcus lysozyme are separation and Extraction ((Schindler C.A.et al (1965) " Purification and properties of lysostaphin:a lyticagent for the Staphylococcus aureus. " Biochem.Biophys.Acta.97:242-250 from imitation staphylococcus culture; IversonO.J.et al (1973) " Studies on lysostaphin, separation and characterization of threeenzymes. " Eur.J.Biochem.38:293-300; Valisena S., F.E.et al (1982) " Purificationand Characterization of three separate bacteriolytic enzymes excreted by S.aureus, S.simulans and S.saprophyticus. " J.Bact.151:636-647; Sugai M., T.et al (1990) " Rapid purification of lysostaphin for analysis of cell-wall proteins. " J.Microb.Meth.12:133-138; Marova I.et al (1993) " Modified simplified method for isolation oflysostaphin from the culture filtrate of Staphylococus staphylolyticus. " FoliaMicrobiol.38:245-252), the not high and heterogeneity of product purity is polluted by thermal source and anaphylactogen easily.The highly purified staphylococcus lysozyme of a large amount of preparations that develops into of genetically engineered and protein separation technology has been opened up new approach.1987, Heath, L.S. etc. expressed staphylococcus lysozyme gene (" Cloning ofthelysostaphin gene of S.simulans biovar staphylolyticus. " Abstr.Ann.Meet.Am.Soc.Microb.H58p 149; " Plasmid encoded lysostaphin endopeptidase gene of S.simulansbiovar staphylolyticus. " FEMS Microb.Lett.44:129-133), Recsei P.A. etc. has cloned complete genome (" Cloning; sequence; and expression of the lysostaphin gene fromStaphylococcus simulans. " Proc.Natl.Acad.Sci.USA 84:1127-1131,1987), Heinrich P., (" The molecular organization of the lysostaphin gene and itssequences repeated in tandem. " Mol.Gen.Genet.209:563-569 such as R., 1987) and United States Patent (USP) U.S.Pat.No.4,931,390 report and disclose complete gene order.At present staphylococcus lysozyme has been realized recombinant expressed in various engineering bacterium such as intestinal bacteria, subtilis, Bacillus sphaericus.The output and the purity of recombinant product all improve a lot, and its physico-chemical property, enzymic activity etc. do not have essential distinction with natural enzyme, for clinical application is laid a good foundation.
Animal experiments such as Dajes studies show that the local dose therapeutically effective eye 0.28% of reorganization staphylococcus lysozyme, nasal cavity using dosages 0.5% such as Kokai-kun, Patron etc. are 5mg/kg to the dosage of animal whole body administration, every day 3 times, therefore effectively the staphylococcus lysozyme therapeutic dose is very high, therefore make up efficient expression strain, exploitation large-scale industrialization technology of preparing is very important to the clinical application of this enzyme.
Nineteen ninety, Bharat Biotech International, Ltd. applied for international monopoly WO01/29201 " Expression of recombinant mature lysostaphin ", this method is directly expressed staphylococcus lysozyme mature peptide gene, expression product is expressed with soluble form in cell, expression amount is 20.2%, protein recovery 8.9mg/L.Chinese patent CN190290A discloses a kind of method of E, coli to express lysostaphin in high efficiency via external secretion, and this method obtains staphylococcus lysozyme 83mg, rate of recovery 27.7mg/L with 3 liters of nutrient solutions through separation and purification.The method of above patent is produced staphylococcus lysozyme and is all existed expression amount low, the problem that yield is low.
In addition, staphylococcus lysozyme is to produce immune response as heterologous protein in the intravital main adverse reaction of machine, causes allergic reaction.Animal pharmacokinetics studies show that this enzyme enters interior 1 hour 95% enzyme of body and degrades.The application of polyoxyethylene glycol (PEG) modification technique is transferred in the modified protein many good characteristics of polyoxyethylene glycol (PEG) and is gone.When polyoxyethylene glycol (PEG) is coupled to the protein molecule surface, can reduce the enzymolysis of pharmaceutical grade protein, avoid very fast elimination in the metabolism of kidney, and pharmaceutical grade protein is difficult for by immune cell recognition.Very big change also takes place in the pharmacokinetic property of PEG class modifier, and the transformation period prolongs.Walsh in 2003 etc. have reported that the PEG of staphylococcus lysozyme modifies, and have adopted the PEG of branch, and PEGization dissolving staphylococcal bacteria half life of enzyme extends to 24 hours.The subject matter of this method is that the PEG of branch cost is very high, is not suitable for the requirement of industrialization.
Prior art expression dissolving staphylococcal bacteria production of enzyme can't satisfy the requirement of mass preparation, and the cost that PEG modifies is very high, therefore is necessary to develop the suitable methods that prepare staphylococcus lysozyme in a large number and reaches modification technique cheaply.
Summary of the invention
The object of the present invention is to provide a kind of expression amount height, yield height, the preparation method of the staphylococcus lysozyme of suitable mass preparation.
Another object of the present invention is to provide a kind of polyethyleneglycol modified cheaply staphylococcus lysozyme.
A further object of the invention provides the preparation method of above-mentioned polyethyleneglycol modified staphylococcus lysozyme.
In order to realize first above-mentioned purpose, the preparation method of the staphylococcus lysozyme that the present invention adopts comprises the steps:
(1), the acquisition of staphylococcus lysozyme gene;
(2), the structure of expression vector and transformed into escherichia coli engineering bacteria: the restriction enzyme site that the staphylococcus lysozyme gene fragment is inserted into the expression vector plasmid, direct then transformed into escherichia coli host bacterium, wherein said expression vector plasmid is a pET serial carrier plasmid;
(3), the screening of positive colony, cultivation, abduction delivering: filter out positive colony and on substratum, cultivate, target protein is expressed through inducing;
(4), the separation of expression product, purifying: at first with colibacillus engineering microorganism collection, broken bacterium, through the centrifugal bacterium liquid supernatant that obtains brokenly, then to broken bacterium liquid supernatant chromatographic technique purifying.
Above-mentioned staphylococcus lysozyme cDNA sequence is known, 246 amino acid whose protein of the total length of encoding.The encoding sequence of staphylococcus lysozyme can derive from gene library, or synthetic according to the full gene of staphylococcus lysozyme cDNA sequence of bibliographical information, can also design primer, obtains by pcr amplification.
As preferably, above-mentioned staphylococcus lysozyme gene order is as described in the SEQ ID NO.1.Here be needs optimization design staphylococcus lysozyme gene order according to escherichia coli high-level expression.
As preferably, above-mentioned pET serial carrier plasmid is one or more among pET-11b, pET-22b, pET-15b, pET-19b, pET-30a-c and the pET-32a-c.
As preferably, on step (2) restriction enzyme site be Nde I/BamHI or Nco I/BamHI site.
As preferably, on step (3) in the positive colony screening be to do resistance screening with penbritin.
As preferably, the temperature of cultivating in the above-mentioned step (3) is controlled at 28 ℃～40 ℃.Preferred again temperature of cultivating is controlled at 30 ℃～35 ℃.
As preferably, the abduction delivering working concentration is that the IPTG of 0.1～1.5mmol/L is as inductor in the above-mentioned step (3).Preferred again abduction delivering working concentration is 0.4～1.0mmol/L.
As preferably, the inductive temperature is controlled at 20 ℃～40 ℃ in the above-mentioned step (3).Preferred again temperature of cultivating is controlled at 25 ℃～30 ℃.
As preferably, chromatographic technique is one or more methods in ion exchange chromatography, sieve chromatography, hydrophobic chromatography and the affinity chromatography in the above-mentioned step (4).
In order to realize second above-mentioned purpose, polyethyleneglycol modified staphylococcus lysozyme of the present invention adopts single-point or multiple spot modification to constitute by staphylococcus lysozyme and polyoxyethylene glycol.
As preferably, above-mentioned polyoxyethylene glycol is a mono methoxy polyethylene glycol.Be selected from mono methoxy polyethylene glycol amber acidic group succinate, mono methoxy polyethylene glycol amber acidic group carbonic ether, the mono methoxy polyethylene glycol aldehyde one or more as preferred again, above-mentioned mono methoxy polyethylene glycol.As most preferably, above-mentioned mono methoxy polyethylene glycol is a mono methoxy polyethylene glycol aldehyde.
As preferably, the molecular weight of above-mentioned polyoxyethylene glycol is 5kDa～100kDa.Preferred again molecular weight is 5kDa～40kDa.Most preferred molecular weight is 20kDa～40kDa.
In order to realize the 3rd above-mentioned purpose, the preparation method of polyethyleneglycol modified staphylococcus lysozyme of the present invention has comprised the preparation process and the polyethyleneglycol modified step of the staphylococcus lysozyme behind the purifying of the staphylococcus lysozyme of above-mentioned first purpose of realization.
The condition of above-mentioned polyethyleneglycol modified reaction is: preparation modification reaction damping fluid, and ionic strength is 5～500mmol/L, the pH value is 4～8; 8～24 hours time, 4～25 ℃ of temperature of reaction.
As preferably, the part by weight of above-mentioned polyoxyethylene glycol and the reaction of staphylococcus lysozyme generation covalent attachment is 0.5～20.Preferred again part by weight is 2～10.
As preferably, the pH value of the damping fluid of above-mentioned polyoxyethylene glycol and the reaction of staphylococcus lysozyme generation covalent attachment is 3～8.Preferred again pH value is 5～7.
As preferably, the temperature of above-mentioned polyoxyethylene glycol and the reaction of staphylococcus lysozyme generation covalent attachment is 4 ℃～37 ℃.Preferred again temperature is 4 ℃～25 ℃.
As preferably, the time of above-mentioned polyoxyethylene glycol and the reaction of staphylococcus lysozyme generation covalent attachment is 4～48 hours.The preferred again time is 16～24 hours.
The preparation method of staphylococcus lysozyme of the present invention, its expressional scheme is simple, and the expression amount height reaches more than 30%, purifying process is simple, the yield height, expression product is solvable, does not need renaturation, directly carry out purifying with chromatography method, purification yield improves greatly, and purification yield reaches 1～3g/L, is fit to mass preparation.
The polyethyleneglycol modified staphylococcus lysozyme of the present invention adopts single-point or multiple spot modification to constitute by staphylococcus lysozyme and polyoxyethylene glycol, polyethyleneglycol modified staphylococcus lysozyme immunogenicity reduces, transformation period prolongs, and be fit to clinical application in the treatment of staphylococcal infections, and the cost of modifying is low.
Description of drawings
Fig. 1: the expression analysis of target protein
M: molecular weight of albumen, from top to down 97.4,66.2,43,30,20.1,14.4kd;
1～3: induce the thalline whole protein; 4: do not induce thalline.
Fig. 2: cation-exchange chromatography collection of illustrative plates.
Fig. 3: hydrophobic chromatography collection of illustrative plates.
Fig. 4: the electrophoretic analysis of purified product
1～3: purify intermediates; 4: purifying Lysostaphin;
M: molecular weight of albumen, from top to down 97.4,66.2,43,30,20.1,14.4kd.
Fig. 5: the RP-HPLC analytical results of purified product.
Fig. 6: polyethyleneglycol modified Lysostaphin electrophoretic analysis
1～3: the Lysostaphin of unmodified; 4: Pegylation Lysostaphin;
M: molecular weight of albumen, from top to down 97.4,66.2,43,30,20.1,14.4kd.
Embodiment
Synthetic, the clone of embodiment 1 staphylococcus lysozyme gene and expressing
Expression vector adopts pET-22b (Invitrogen), and the host bacterium is e. coli bl21 (DE3).
The Lysostaphin gene order (AX828346) of the Staphylococcus simulans that announces according to Genebank and the staphylococcus lysozyme gene order of patent WO03074689, needs optimization design staphylococcus lysozyme gene order according to escherichia coli high-level expression, see SEQ ID NO.1, the aminoacid sequence of gene encoding production is seen SEQ ID NO.2.0 section complementary oligonucleotide of Synthetic 2, and hold at 5 ' end and 3 ' and to draw respectively as restriction enzyme site Nde I, BamH I, press the ordinary method of molecular cloning, at first handle 30min for 37 ℃ with T4 phage polynucleotide kinase, each oligonucleotide fragment of phosphorylation mixes to wait mole, 94 ℃ of sex change 5min, 65 ℃ of annealing 10min immediately, add the T4 ligase enzyme then, 16 ℃ of connections are spent the night.Getting the connection product increases with following primer:
Primer 1:5 '--TATACATATGGCTGCAACACATGAACA
Primer 2: 3 '--TCTGGATCCTCAGTTACTTTATAGTTCCCCAAA
Amplification condition: 95 ℃, 30s; 55 ℃, 30s; 70 ℃, 60s, cycle index: 30 times.10min is extended in last 70 ℃ of insulations.Digest the PCR product of described staphylococcus lysozyme with Nde I and BamH I.
The pET-22b plasmid reclaims big fragment with Nde I, BamH I double digestion, is connected with synthetic staphylococcus lysozyme gene fragment, and 20 μ L reaction system gene fragments and the big segmental ratio of carrier are 10: 1, add T
4Dna ligase 300 units, 15 ℃ of connections are spent the night, and get 10 μ L and connect product direct transformed into escherichia coli host bacterium BL21 (DE3) competent cell, coat the amicillin resistance flat board, and 37 ℃ of overnight incubation obtain engineering bacteria through further screening.Penbritin is done resistance screening, obtains positive colony.Extract plasmid, identify with restriction enzyme.Positive transformant carries out sequential analysis with universal primer, and cloned sequence and implementation sequence are in full accord as a result.
The inoculation positive colony is cultivated, and induces through the IPTG of 1mmol/L, the results are shown in Figure 1.
Adopt substratum (0.5%Yeast Extract, 1.0%Peptone, 0.5%NaCl, 0.05mg/LAmp pH7.4) has studied condition of different temperatures and has induced influence to the product expression status.Inoculum size: 5% (the seed liquor volume accounts for the ratio of culture volume), culture temperature is 37 ℃ before inducing, and adopts IPTG to induce dosage 1mmol/L.Cultivate back 4 temperature and induce, the results are shown in following table.
| Inducing temperature | Expression amount | Target protein supernatant % | Target |
| 20℃ | 20％ | 90％ | 10％ |
| 25℃ | 30％ | 90％ | 10％ |
| 30℃ | 30％ | 65％ | 35％ |
| 37℃ | 30％ | 50％ | 50％ |
Sum up: add behind the inductor 25 ℃ and cultivated 4 hours, expression amount can reach 30%, and target protein mainly is expressed in brokenly in the bacterium supernatant, if prolong expression time, as expressed 8 hours, then target protein surpasses in 80% the precipitation behind broken bacterium, after therefore preferred condition is 37 ℃ of cultivations, 25 ℃ of abduction deliverings 4 hours, target protein mainly in the supernatant, help separation and purification behind broken bacterium.
Separation, the purifying of embodiment 3 expression products
(0.5mM EDTA is pH8.0) according to the ratio suspension wet thallus of 20ml/g for 40mM Tris-Cl, 02M NaCl with the bacterial cell disruption damping fluid.The broken thalline (220V, 20khz, 900W, ultrasonication under the ice-water bath condition) of supersonic method.
To break bacterium liquid and carry out centrifugal (10000g, 30min, 4 ℃), remove precipitation, collect supernatant with high speed centrifugation.The broken bacterium supernatant liquor ion exchange chromatography separation and purification of collecting, its process makes its specific conductivity less than 5mS/cm for being that 8.5 20mmol/L Tris.CL damping fluid dilutes with pH value.With same damping fluid balance chromatography column
, chromatography gel can be selected CM-Sepharose Fast Flow, SP-Sepharose Fast Flow, Mono-S (Amersham Pharmacia) for use.Be washed till baseline with level pad behind the last sample, (0.01～0.5mol/L NaCL, 20mM Tris-C1 pH8.0) carry out gradient elution, occur target protein when NaCL concentration reaches 30%, collect the target protein peak, and the chromatography collection of illustrative plates is seen Fig. 2 with damping fluid.The protein sample purity that wash-out comes out is at 85-90%.(NH is added at ion-exchange target protein peak
4)
2SO
4Making final concentration is 0.6～0.8mol/L, hydrophobic chromatography post (XK26/20) buffer system A (20mM Tris-Cl, pH8.5,0.6M (NH
4)
2SO
4) balance, organophilic gel can be selected Phenyl-Sepharose Fast Flow, Phenyl-Sepharose High Perforance and Butyl-Sepharose Fast Flow for use, and flow velocity 10ml/min uses buffer B (20mM Tris-Cl behind the last sample; PH8.5) wash and drag by reducing the salt concn gradient, collect the target protein peak, the hydrophobic chromatography collection of illustrative plates is seen Fig. 3.Target protein electrophoresis purity 95%, analytical results is seen Fig. 4.
The hydrophobic chromatography peak that obtains is through Sephadex G-25 desalination, and balance liquid is 20mmol/L PB, pH7.4.Be the staphylococcus lysozyme of polishing purification after the sample desalination.
The enzyme activity assay of embodiment 4 purified products
Enzyme activity assay adopts turbidimetry, method (Cloning with reference to Piotr Szweda etc., Expression, and Purfication of the Staphylococcus simulans Lysostaphin Using theIntein-Chitin-Binding Domain (CBD) System (Protein), being tried bacterium is streptococcus aureus Staphylococcus aureus.The activatory streptococcus aureus in the LB substratum 37 ℃ to be cultured to OD620nm be 0.25, add an amount of purifying lysostaphin (10 μ L, about 1 μ g), measure the absorption value of OD620nm behind 37 ℃ of insulation 10min.Unit of enzyme activity is defined as the following 37 ℃ of insulation 10min of pH7.5 condition, is that the 0.25 0.125 required enzyme amount of reducing to is 1 a unit with 6ml streptococcus aureus suspension OD620nm.Dissolving staphylococcal bacteria specific enzyme activity of the present invention reaches 412U/mg.
The purity check of embodiment 5 purified products
The purity of purifying lysostaphin adopts the RP-HPLC method further to analyze, and analytical column is the Vydac 214TP5415 of Alltech company, 150mmX4.6mm, 5 μ m, mobile phase A: 0.1%TFA, ACN-H
2O (5: 95); Mobile phase B: 0.1%TFA, ACN-H
2O (95: 5), flow velocity 0.8ml/min detects wavelength 214nm, and analytical procedure sees the following form:
| Step | Time(min) | B (％) | Max Press(bar) |
| 1 | 0 | 20 | 200 |
| 2 | 10 | 20 | 200 |
| 3 | 30 | 40 | 200 |
| Step | Time(min) | B (％) | Max Press(bar) |
| 4 | 40 | 40 | 200 |
| 5 | 65 | 80 | 200 |
Sample purity reaches 99%, and analytical results is seen Fig. 5.
The staphylococcus lysozyme (LSS) of purifying is dissolved in the phosphate buffered saline buffer of pH 8.0,100mM, in staphylococcus lysozyme: after the ratio of mono methoxy polyethylene glycol aldehyde=1: 5 (w/w) adds dissolving, add NaCNBH
3Making its final concentration is 8mM, 2～8 ℃ of reactions of refrigerator 16～24 hours.Modification reaction finishes the back and adopts molecular sieve column (Sephacryl-200) to remove the LSS and the free polyoxyethylene glycol of unmodified.With pH 8.0,20mM PB is elutriant, flow velocity 5ml/min, and collecting first protein peak is the modified protein peak, electrophoretic analysis the results are shown in Figure 6.
Sequence table
＜110〉Hangzhou Biodoor Biotechnology Co., Ltd.
＜120〉preparation method of a kind of staphylococcus lysozyme and derivative thereof
<160>2
<210>1
<211>757
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)..(741)
<220>
<221>misc feature
＜223〉Lysostaphin encoding sequence
<400>1
ATGGCTGCAA CACATGAACA TTCAGCACAA TGGTTGAATA ATTACAAAAA 50
AGGATATGGT TACGGTCCTT ATCCATTAGG TATAAATGGC GGTATGCACT 100
ACGGAGTTGA TTTTTTTATG AATATTGGAA CACCAGTAAA AGCTATTTCA 150
AGCGGAAAAA TAGTTGAAGC TGGTTGGAGT AATTACGGAG GAGGTAATCA 200
AATAGGTCTT ATTGAAAATG ATGGAGTGCA TAGACAATGG TATATGCATC 250
TAAGTAAATA TAATGTTAAA GTAGGAGATT ATGTCAAAGC TGGTCAAATA 300
ATCGGTTGGT CTGGAAGCAC TGGTTATTCT ACAGCACCAC ATTTACACTT 350
CCAAAGAATG GTTAATTCAT TTTCAAATTC AACTGCCCAA GATCCAATGC 400
CTTTCTTAAA GAGCGCAGGA TATGGAAAAG CAGGTGGTAC AGTAACTCCA 450
ACGCCGAATA CAGGTTGGAA AACAAACAAA TATGGCACAC TATATAAATC 500
AGAGTCAGCT AGCTTCACAC CTAATACAGA TATAATAACA AGAACGACTG 550
GTCCATTTAG AAGCATGCCG CAGTCAGGAG TCTTAAAAGC AGGTCAAACA 600
ATTCATTATG ATGAAGTGAT GAAACAAGAC GGTCATGTTT GGGTAGGTTA 650
TACAGGTAAC AGTGGCCAAC GTATTTACTT GCCTGTAAGA ACATGGAATA 700
AATCTACTAA TACTTTAGGT GTTCTTTGGG GAACTATAAA GTAACTGAGG 750
ATCCAGA 757
<210>2
<211>247
<212>PRT
＜213〉intestinal bacteria (Escherichia Coli)
<400>2
Met Ala Ala The His Glu His Ser Ala Gln Trp Leu Asn Asn Tyr Lys Lys Gly Tyr Gly
1 5 10 15 20
Tyr Gly Pro Tyr Pro Leu Gly Ile Asn Gly Gly Met His Tyr Gly Val Asp Phe Phe Met
25 30 35 40
Asn Ile Gly The Pro Val Lys Ala Ile Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser
45 50 55 60
Asn Tyr Gly Gly Gly Asn Gln Ile Gly Leu Ile Glu Asn Asp Gly Val His Arg Gln Trp
65 70 75 80
Tyr Met His Leu Ser Lys Tyr Asn Val Lys Val Gly Asp Tyr Val Lys Ala Gly Gln Ile
85 90 95 100
Ile Gly Trp Ser Gly Ser The Gly Tyr Ser The Ala Pro His Leu His Phe Gln Arg Met
105 110 115 120
Val Asn Ser Phe Ser Asn Ser The Ala Gln Asp Pro Met Pro Phe Leu Lys Ser Ala Gly
125 130 135 140
Tyr Gly Lys Ala Gly Gly The Val The Pro The Pro Asn The Gly Trp Lys The Asn Lys
145 150 155 160
Tyr G1y The Leu Tyr Lys Ser Glu Ser Ala Ser Phe The Pro Asn The Asp Ile Ile The
165 170 175 180
Arg The The Gly Pro Phe Arg Ser Met Pro Gln Ser Gly Val Leu Lys Ala Gly Gln The
185 190 195 200
Ile His Tyr Asp Glu Val Met Lys Gln Asp Gly His Val Trp Val Gly Tyr The Gly Asn
205 210 215 220
Ser Gly Gln Arg Ile Tyr Leu Pro Val Arg The Trp Asn Lys Ser The Asn The Leu Gly
225 230 235 240
Val Leu Trp Gly The Ile Lys
245
Claims (10)
1. the preparation method of staphylococcus lysozyme is characterized in that comprising the steps:
(1), the design of staphylococcus lysozyme gene is with synthetic, the staphylococcus lysozyme gene order is as described in the SEQ IDNO.1;
(2), the structure and the transformed into escherichia coli engineering bacteria of expression vector: the staphylococcus lysozyme gene fragment is inserted into the restriction enzyme site of expression vector plasmid, direct transformed into escherichia coli host bacterium then, wherein said expression vector plasmid is pET-22b;
(3), the screening of positive colony, cultivation, abduction delivering: filter out positive colony and on substratum, cultivate, target protein is expressed through inducing;
(4), the separation of expression product, purifying: with colibacillus engineering microorganism collection, broken bacterium, through the centrifugal bacterium liquid supernatant that obtains brokenly, then to broken bacterium liquid supernatant chromatographic technique purifying.
2. the preparation method of staphylococcus lysozyme according to claim 1, the acquisition that it is characterized in that the staphylococcus lysozyme gene is synthetic by full gene.
3. the preparation method of staphylococcus lysozyme according to claim 1 is characterized in that the temperature of cultivating in the step (3) is controlled at 28 ℃～40 ℃.
4. the preparation method of staphylococcus lysozyme according to claim 1 is characterized in that inducing temperature is controlled at 20 ℃-37 ℃ in the step (3).
5. the preparation method of staphylococcus lysozyme according to claim 1 is characterized in that the middle chromatographic technique of step (4) is one or more methods in ion exchange chromatography, sieve chromatography, hydrophobic chromatography and the affinity chromatography.
6. the preparation method of polyethyleneglycol modified staphylococcus lysozyme, it is characterized in that: this method comprises the preparation process and the polyethyleneglycol modified step of the staphylococcus lysozyme behind the purifying of staphylococcus lysozyme as claimed in claim 1, and staphylococcus lysozyme and polyoxyethylene glycol adopt single-point or multiple spot to modify formation.
7. the preparation method of polyethyleneglycol modified staphylococcus lysozyme according to claim 6 is characterized in that the part by weight of polyoxyethylene glycol and staphylococcus lysozyme generation covalent attachment reaction is 0.5～20.
8. according to the preparation method of claim 6 or 7 described polyethyleneglycol modified staphylococcus lysozymes, the pH value that it is characterized in that the damping fluid of polyoxyethylene glycol and staphylococcus lysozyme generation covalent attachment reaction is 4～8, the temperature of reaction is 2 ℃～37 ℃, and the time of reaction is 4～48 hours.
9. according to the preparation method of claim 6 or 7 described polyethyleneglycol modified staphylococcus lysozymes, it is characterized in that described polyoxyethylene glycol is selected from one or more in mono methoxy polyethylene glycol amber acidic group succinate, mono methoxy polyethylene glycol amber acidic group carbonic ether and the mono methoxy polyethylene glycol aldehyde.
10. according to the preparation method of claim 6 or 7 described polyethyleneglycol modified staphylococcus lysozymes, the molecular weight that it is characterized in that polyoxyethylene glycol is 5kDa～100kDa.
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| CN103131687A (en) * | 2013-02-04 | 2013-06-05 | 杭州北望生物技术有限公司 | Polyethylene glycol covalent modification lysostaphin and prepared method and application thereof |
| US11155801B2 (en) * | 2013-08-06 | 2021-10-26 | Trustees Of Dartmouth College | Unglycosylated lysostaphin variant protein |
| CN111560048B (en) * | 2020-06-03 | 2021-08-13 | 成都正能生物技术有限责任公司 | Method and kit for extracting total protein of staphylococcus aureus |
| CN114540385B (en) * | 2022-03-21 | 2023-06-23 | 齐鲁工业大学 | Method for producing lysostaphin by using escherichia coli |
| CN115873834B (en) * | 2022-08-26 | 2024-06-04 | 山东大学 | Lysostaphin variant and preparation method and application thereof |
| CN115976158B (en) * | 2023-03-16 | 2023-07-04 | 百葵锐(天津)生物科技有限公司 | Method for measuring enzyme activity of lyase |
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